TW201022439A - Novel strain of bacillus amyloliquefaciens and its use - Google Patents

Abstract

An isolated Bacillus amyloliquefaciens Ba-BPD1 having a BCRC Accession No. 910395 is provided. This novel strain has an unique 16S ribosomal RNA sequenced as SEQ ID NO: 1 and produces amylase, protease, cellulase, lipase and fibrinolytic enzyme to show their biodegradation capacities. Further, B. amyloliquefaciens Ba-BPD1 produces the antibiotic substances, such as iturin, fengycin and surfactin, and has antimicrobial capacity for inhibiting the fungal or bacterial growth. In conclusion, the novel strain of Bacillus amyloliquefaciens Ba-BPD1 and its products can be applied in agriculture, wastewater treatment, food industry and chemical industry.

Description

201022439 IX. Description of the invention: [Technical field to which the invention pertains] This case relates to a novel liquefied Bacillus amyloliquefaciens strain, especially in the case of a novel Bacillus amyloliquefaciens strain BaciHus amyloliquefaciens Ba-BPD1 or a mutant thereof, Used to produce a variety of enzymes, a variety of anti-biomass and biosurfactants. [Prior Art] φ Microorganisms and their products have been widely used to enhance human life, such as in food, alcohol, pharmaceuticals, chemical industry, and agriculture. These applications significantly reduce production and/or processing costs as well as meet human needs. Some microorganisms produce enzymes to break down giant molecules. For example, Yarrowia (Kzmwk/fine 〇〇·〇〇 produces lipolytic enzymes (lipases) to degrade the chemical oxygen demand (COD) of olive mill wastewater treatment (15); Pseudomonas aeruginosa CPsewcfo/womw izerwgenasa) produces alkaline protease to hydrolyze the main protein solid waste in the leather manufacturing industry - animal meat quality 〇 4); and soil mites (乂甲 er#//w5 ❹ produces carboxymethylase (carboxymethyl) Cellulase, CMCase) is used to biodegrade lignocellul〇sic waste (7). However, these microorganisms have been shown to function as a major enzyme to decompose a substrate. When dealing with more complex components of municipal wastewater, Microorganisms are necessary and inevitable to degrade a variety of macromolecules. This will increase costs, reduce economic efficiency, and the processing will be more complicated. Furthermore, these added microorganisms may compete with each other for growth advantages. The ability of the enzyme can only be applied to one field. The economic benefit of this microorganism is more than that of other enzymes. Microorganisms are low in the field. 5 201022439 In addition to biodegrading organic waste, microorganisms can also produce antibiotics to antagonize other fungi or bacteria. In general, antibiotics are produced or extracted from fungi and used in medicine. Fruits and animals are also exposed to fungal or bacterial infections during their maturation and growth. Traditional fungicides, bactericides and chemical synthetic agents do not inhibit fungal and bacterial infections, but also harm the human body and the environment. If bacteria are found to produce biological properties Antibiotics to inhibit the growth of fungi or bacteria, this strain will benefit agriculture and animal husbandry. Recently, 'a unique amphiphilic biological compound produced by microorganisms _ biosurfactant, already in organic and metal contaminated areas The restoration shows a variety of potential applications (3). Biosurfactants reduce surface tension, stabilize emulsification and promote foam formation, and are generally non-toxic and biodegradable. Biosurfactants are classified as glycolipids and lipids. Protein (lip0pr〇tein), in which lipoproteins include Ituri, (surfactin) and fengyin (fengydn) produced by the bacillus net (10) sp.). Microorganisms that produce biosurfactants play an important role in the bioremediation of hydrocarbon-contaminated areas. Biosurfactants can also be used to promote oil recovery and are considered to have other applications in environmental protection. Other applications include herbicide and insecticide formulations, detergents, health and cosmetics, pulp and paper, coal, textiles, pottery processing and food industry, uranium processing and peat mechanical dewatering. Therefore, if microorganisms are found to produce a variety of enzymes and molecules, this microorganism will benefit human life and economy. In view of the shortcomings in the prior art, the applicant of this case, after careful experimentation and research, and a spirit of perseverance, finally conceived the case of the novel "Newly liquefied Bacillus amyloliquefaciens strain and its application", selected and fine Ba -BPDl has the ability to produce the above enzymes, anti-biomass and biosurfactant compared to other microorganisms 'can overcome the prior art's non-201022439 foot' following the brief description of this case. SUMMARY OF THE INVENTION The present invention proposes that the selected strain 'has a specific 16S nuclear listener _>somal RNA, fRNA) sequence 'minute _ liquid tilt (four) scale g (heart chest / 〇 / _ plus ▲ Ba_BPD1), The patent microbiology deposit number of its Food Industry Development Research Institute is BCRC 910395. This new strain produces specific and beneficial enzymes such as lipolytic enzymes (4) decomposing lipids, and decomposing enzymes to hydrolyze starch, cellulolytic enzymes, hydrolyzed cellulose, and proteolytic enzymes to hydrolyze proteins. - New organic liquefied Bacillus licheniformis (5-like Ba-BPDl) is used in the treatment of sewage, water pipeline systems, animal feed, and kitchen waste to enhance organic matter in sewage. Material decomposition and/or water quality, quality and efficiency of the waste treatment process. Therefore, the novel strain and its enzyme can be made into a detergent and applied to decontamination and food manufacturing. Furthermore, the novel Bacillus amyloliquefaciens producing a variety of enzymes is used in the agricultural ginseng industry, including green storage inoculants such as 6 1110 (^1 post 8), livestock manure treatment and animal feed probiotics (^ Direct Fed Microbials ). Furthermore, due to its enzymatic activity of decomposing macromolecules into simple organic molecules, the selected 5 can promote plant growth. In addition, thrombolytic enzymes produced by ugly Ba-BPD1 can hydrolyze thrombus' to reduce the amount of blood clots in the blood, prevent and treat cardiovascular diseases, embolism, arteriosclerosis, endometriosis and cancer. Therefore, thrombolytic enzymes can promote the health of humans and animals. In addition, the isolated and Ba-BPD1 produce anti-biomass 7 201022439 (including, subtilisin, surfactant and amylin) and surfactants. In particular, it is a subtilisin and its homologue. Irisin, surface active substances, and elemental substances are fat-like and are beneficial for the prevention and treatment of fungal and/or bacterial infections, which are plants, animals and fruits. In addition, the production of biosurfactants, including activin, iturin and abundance, inhibits the growth of plant pathogens and animal pathogens and has the potential to be resistant to biomass production. Another object of the present invention is to know that the novel strain B. amyloliquefaciens • Ba-BPD1 and/or the antibiotic produced by the strain is used as an antifungal agent to inhibit the growth of at least one fungal genus belonging to the following group The composition group includes: Boifyiis, CoHeioirichum, Rhizoctonia (10) Z2〇ci (wic〇, Pwsariwm, Sc/eroriwm) , Altemaria, Phytophthora, Aspergillus, PeniciWum, jPesialotiopsis, and Botryodiplodia ° The fungal infection is derived from the following group of fungi: Botrytis elliptica, Botrytis cinerea, G. mellifera (G/omere//c ckgM/ato), banana anthrax Pathogens (CW/eioiric/mm mwsae), CoUetotrichum gloeo^orioides, Rhizoctonia solani, Fusarium oxysporum f. sp. pisi, tomato cells Fungus (Fusarium oxysporum f. sp. fycopersici), 籣花镰Fusarium solani, Fusarium solani, Sclerotium rolfsii Saccardo, Akernaria maii, Phytophthora capsid, foreign ticket, AspergiUus niger, Penicillium italicum, Pestalotiopsis eugeniae, and Botryodiplodia theobromae. Another novel strain B. amyloliquefaciens Ba- The anti-biomass produced by the BPD1 anti-/or collusion strain is used as an antibacterial agent for inhibiting the growth of at least one of the genus Bacteroides belonging to the following group consisting of: Escherichia, Acidophilus Genus (Jcii/ovorox), Agrobacterium η Μ Μ, B. burdori (Bwr/io/i/en'fl), genus Pythium (Pseudomonas, Pseudomonas) Ralstonia, Xcmthomonas, Bacillus (this continent (10)) and Salmonella (Salmme (4)). ❹ wherein the bacterial infection is caused by the following group of bacteria: Erwinia chrysanthemi and Erwinia carotovom subsp. caroiovom, bacterium melon-like spot bacteria (▲zV/ovorax avewae subsp. czYrw/"), Carcinoma sinensis (iwme/(10)>like), Carnationaceae flower, Braholderia caryophylU, Enter〇bact〇rc/ofleeae, Peach bacterium ^•«容ae), bacterial wilt 5O/imacearwm), citrus canker (Xim^OWIOWas like 0 〇 〇 p also pV czW), Jiake plant bacterial spot bacteria (yi (10) 决 (10) (10) like vesicatoria) , Nico's black rot fungus (xanih〇mmas (10), which is pv compestfis), rice chlorophyllin (^anth〇monas 〇fyzae pV 〇ryzaey, and Bacillus cereus anti-Salmonella). The selected 5. am;;/〇/^rM^/^ewBa_BPD1 is used as an antibacterial agent for inhibiting the growth of fungi and/or bacteria, and the antifungal agent inhibits the growth of fungi belonging to the following group: Livureum, Botrytis cinerea, lemon anthracnose, Anthracnose, sweet persimmon anthracnose, Rhizoctonia solani, Peony cyanobacteria, Tomato bacillus, Lotus bacillus, Litchi licheniformis, Liriomyza sativa, Apple brown spot, Sweet pepper , onion black fungus, citrus green mold, lotus leaf rot fungus and lemon fruit 201022439. Antibacterial agent inhibits bacteria belonging to the following group of groups ^, dry bacteria, melon bacterial spot bacteria, cancer bacteria, Dianthus Branch flower: sputum = field; sputum-based rot fungus, peony bacterial spotted bacterium, green bacterium, citrus

Therapy is carried out by the growth of pathogens of livestock and food, and the prevention or/or treatment of diseases or plants. In addition, by the characteristics of surfactants are widely used in cleaning two = food, mouth, music, petrochemical, agriculture and environmental protection. According to the above concept, the selected application forms are all liquid culture medium (wh〇le br〇th, supernatant, wettable powder (wettable P〇wder), granules (known coffee (6), water) Dispersible granules are arbitrarily granules, water suspensions (suspensi〇n c_n_ 〇r fl_we __te) and microencapsulation forms. The purpose of this case is to propose a patent microbial deposit number bcrc 910395.

Screening process of Phytophthora sinensis (IV) with a specific (10) sequence (SEQ ID NO: 1) 另一 Another object of the present invention is to include a composition having a liquefaction with the patented microbial accession number BCRC 910395; Bacillus licheniformis. [Embodiment] The "inventive Bacillus amyloliquefaciens strain and its application" proposed in the present invention will be fully understood by the following examples, so that those skilled in the art can complete it. However, the implementation of the present invention is not limited to the following. The embodiments are intended to be limited to the scope of the invention, and those skilled in the art can still practice other embodiments in accordance with the spirit of the embodiments disclosed herein. Example 1: New liquefied starch bud spores The novel liquefied Bacillus amyloliquefaciens (and Ba-BPD1) were screened by the inventors from the soil of Lishan, Taichung County, and further cultured, identified and preserved. When 5. am less Ba-BPD1 was cultured, a single colony was inoculated and cultured overnight in 6 ml of LB medium (Luria-Bertani, Miller, Difco). The cultured bacterial solution was inoculated to 500 ml of LB medium at a ratio of 1/100, and cultured at 30 ° C, 150 rpm for 6 days. ❿ In addition, 'B-BPD1 has a specific 16S ribosomal RNA sequence compared to other bacteria', and part of its 16S ribosomal sequence is sequenced and will be published on the National Biotechnology Information Center website on December 31, 2009. (National Center for Biotechnology Information, NCBI, http://www.ncbi.nlm.gov/Genbank/) 'The GenBank serial number is named EF137183. This portion of the 16S ribosomal sequence (SEQ Π) NO: l) as described later becomes a special and important feature of ^^myloUquefaciens Ba-BPD1. And Ba-BPDl was deposited in the Financial Industry Development Institute of the People's Republic of China on August 15, 1997. The patent microbiological registration number is BCRC 910395. Example 2: and (10) iv/o/iVyifg/iia'g/is Ba_BPDl produced a powdery vocabulary Xin in order to prove that <3 of 3; /〇%^ is a coffee like 8&-:61>01 can produce a temple The powder decomposing enzyme (amylase) is hydrolyzed starch, and the amylase hydrolysis test is as follows. A single colony of 5. Ba-BPD1 was picked from a nutrient agar (NA) plate and mixed with 50 μl of sterile water as a bacterial solution. Then, the 5 μμ1 bacteria were dropped on a 1 cm diameter filter paper and placed on a yeast extract-soluble starch agar (YSA) plate containing 1.〇〇/0 yeast. Extract 201022439, 1.0% soluble starch and 1.5% amaranth). The YSA medium was incubated at 30 ° C for 2 to 3 days. After incubation, 'YSA medium was covered with 3 to 4 ml moth (containing 0.3% (w/v) moth and 3% (w/v) potassium iodine), and measured in Bamin B-like in 5 minutes. Colony size and size of the dissolution zone (8) earzone. The medium turned blue-black to indicate that the powder was not hydrolyzed, however, the dissolution circle around the colon indicated that the starch was hydrolyzed. The results of the three replicates showed that the colony diameter and the dissolution circle size were 1.57 cm and 2.81 cm, respectively.

Ito et al. (1998) also confirmed that basophilic Bacillus subtilis produces alkaline extracellular cleansing enzymes, including amylase, which are used in potent detergents and automatic dishwashing detergents to break down starch in sewage (12). ). Therefore, the amylolytic enzyme produced by the company can be used to hydrolyze sewage, waste, starch in agriculture and food. f Example 3: and flinv/o/iVyKg/iic/giis Ba-RPDl produced | White Decomposition - Prime To demonstrate and Ba-BPD1 production of proteolytic enzyme (protein S#) hydrolyzed protein 'proteolytic test' is described below. And (10)

The bacterial solution of Ba-BPD1 was prepared as described in Example 2. 5 〇 μ1 of the bacteria was dropped on a j cm diameter filter paper and placed in a skim milk agar (SMA) plate containing 1.5 ° /. skimmed milk powder, 1.3% nutrient solution (nutrientbr〇th NB) and 丨5〇/〇洋菜)上〉. The SMA medium was cultured at 3 ° C for 2 to 3 days, and measured.

Colony size and dissolution circle size of Ba-BPD1. The protein in the Lorez non-skimmed milk around the colony is hydrolyzed by bacteria. The results of the three replicates showed that the diameter of the bacteria and the size of the dissolution circle were 177 cm and 3.61 cm, respectively.

Kumar et al. (2008) found that P. aeruginosa produces alkaline proteases to hydrolyze protein solid waste produced by skin factories (4). In addition, Dr〇uin et al. (10) 8) Lichens have a proteolytic activity (5). In the third embodiment, and

The proteolytic enzyme produced by Ba-BPDl can also be used for proteolysis of sewage, waste, agriculture and food industries, and can be made into a detergent or laundry detergent component. Example 4: and the lycopene decomposed by the flmvtofejrtfg/iicfewsBa-BPD1 In order to prove that Ba-BPD1 produces cellulolytic enzyme (cellulase) hydrolyzed cellulose, the cellulose hydrolysis test is as follows. and

Ba-BPD1 bacterial solution was prepared as described in Example 2. 50 μM bacteria was dropped on a 1 cm diameter filter paper and placed in Mandel-Reese (M_R) agaric culture medium (containing 1.0% methyl alcohol cellulose (CMC), 0.1% digested protein) (peptone), 0.03% urea, 0.14% (NH4)2S〇4, 0.2% ΚΗ2Ρ04, 0.04% CaCl2.H20, 0.03% MgS04.7H20, 5x1α4% FeS〇4.7H20, 1.4xl〇-3% ZnS04.7H20, 1.6X10·3% MnS04.4H20, 2xl (TV〇CoCV6H2〇 and 1.5% agar, adjusted to pH 6.0 and sterilized) (16). This MR vegetable culture medium is cultured in 30T for 2 days, 3 to 4 Ml 0.1% Congo Red is overlaid on MR Ocean's medium for 30 minutes, and then unbound Congo red 'Congo red is washed with 1 Μ sodium chloride solution to form agglomerate or colloid φ by hydrogen bonding. (coll〇id), combined with cellulose, and the formation of the dissolution ring indicates that the cellulose did not bind to Congo red. The results of the three repetitions showed that the dissolution circle formed by B. Ba-BPD1 was 2.3 cm in diameter.

Alam (2008) confirmed that the Harz wood emblem (THc/ioi/erma ZiarziimMm) produces fibrinolytic enzymes for hydrolyzing cellulose (2) in the biological conversion of sewage sludge. Sangave and Pandit also published in 2006 a pretreatment step for the biodegradation activity of cellulase for distillate wastewater to convert cellulose into simple biomolecules (called. Since the main component of MR acacia medium is carboxymethyl fiber) It is quite obvious that Ba-BPD1 can produce cellulolytic enzymes to digest cellulose 13 201022439 and to show transparent rings during digestion. Therefore, due to the production and hydrolysis activity of cellulolytic enzymes, ugly treatment of wastewater Has a significant economic value on cellulose. Novel strains 5

Ba-BPD1 produces cellulolytic enzymes that hydrolyze waste-treated cellulose.掩 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Lipase) to break down lipids, lipolytic enzyme hydrolysis test as described below. First, a single colony of indica-Ba-BPD1 was inoculated into 5 ml of nutrient solution, and cultured at 3 ° C, 150 rpm for 1 day. Then, '5 μl of cultured bacteria was dropped on Rhodamine B agar plate (containing 1% olive oil, 0.001% scented red sorghum and 1.5% nutritious agar) and cultured at 3 °C. 7 days. As a dye, the scent of red scent is embedded in lipids and is used as a fluorescent marker in biotechnological applications such as fluorescent microscopes. Dissolving hydrazine means that the lipid is hydrolyzed' and rose begonia cannot be embedded in the lipid. Therefore, as a result of the experiment, it was found that Ba-BPD1 showed a fluorescent colony, and the diameter of the dissolution circle around the colony was 0.6 cm. φ et al., 2007, published: The lipolytic enzyme produced by Bacillus subtilis appears to be composed of the sewage composition of the decomposed olive grinding mill, triacetin, polysorbate 80 (Tween 80) and whey (whey). Etc. lipolytic activity (8). Even, immobilized lipase is used for water/1 solution of sewage with high oil and oil concentrations. Therefore, the lipolytic enzyme produced by Ba-BPD1 can be applied to lipid decomposition in sewage, waste, agriculture and food industries. EXAMPLES ························································································································ Fibrin alone or in combination with its 201022439 material (eg fibrinogen and thrombin) has been used in the bio-skeleton of stem cells or primary cells to regenerate adipose tissue, heart-grade woven, cartilage, liver , nerve tissue, eye tissue, skin, tendons and ligaments, and show significant potential for tissue regeneration and wound healing (1). However, if fibrin is in the blood vessels or heart: agglutination is a fibrin clot or thrombus, which can lead to cardiovascular disease or death (10). Bacillus subtilis strains have been shown to produce gold plug decomposing enzymes for decomposing thrombus by forming active plasmin from plasminogen or by direct fibrinolysis. Therefore, thrombolytic enzymes produced by micro-organisms are important in tissue regeneration, wound healing, and life-saving first aid. In the present invention, a single colony inoculated with 5. am less like Ba-BPD1 was cultured at 5 ° C for 12 hours at 30 °C. After centrifuging 100 μl of the bacterial solution in 5 ml of the nutrient solution, 20 μl of the supernatant was dropped into a shallow hole of a fibrin agar plate previously excavated with a tip, and the fibrin agar. The medium was cultured at 37 ° C for 12 hours, and the formation of a dissolution circle was observed. The results showed that the dissolution circle diameter was 1.8 cm' confirmed and (10) ground/per/(10)·ca Ba_Bpm had the ability to produce thrombolytic enzyme φ to hydrolyze thrombus, and involved, for example, thrombosis, arteriosclerosis, intrauterine Endometrisis and the pathological state of cancer. Example 7 ·················································································································· Bioactive microbial secondary metabolites 'and show attractive antibiotics % of the genus Ishigamycin A produced by the genus of the genus of the genus (the iturin 々 and _ molecules in the pathogenic really fine as the dry simmering oil "〇" The surface of the cell membrane forms a complex, so that the size of the ion channel changes to the osmotic pressure of the membrane of 201022439, and further causes the mycelium of the pathogenic fungus to decompose and inhibit spore germination. Thereby, the inhibitory effect of the plant pathogenic bacteria is achieved. Therefore, italin A and Bacillus subtilis are used for feed and/or food preservation, animal and plant prevention and/or treatment, and can be used for industrial, agricultural, environmental biodegradation and removal of surfactants (or organisms). Surfactants), as well as antibiotics for animal and/or plant infections (17). Please refer to FIG. 1 , which is a liquid chromatography/time-to-flight-mass spectrometry (LC) according to the seventh embodiment of the present invention, and the liquid chromatography/time-to-flight-mass spectrometry (LC) produced by Ba-BPD1. /TOF-φ MS) Analysis of the spectrum. In Figure 1, the molecular weights of the isoformin A homologs (A2 to A8) were identified as 1043, 1057, 1065, 1079, 1095 and 1119 Da, these isoforms A homologs and flmj/o / sentence Ba-BPDl can be applied to the food industry and agriculture. Amygdalin is another important role played by Bacillus subtilis (and bioactive lipopeptides and antifungal substances produced by ZCZ7/WS, and against cucurbit powdery mildew) (4'18). Like Isein A, amylin is also used in feed and/or food preservation as a surfactant (or biosurfactant) for biodegradation and removal of biotechnology, agriculture, and environmental, and as an animal and / or plant-infected antibiotics. Please refer to Fig. 2' for the LC/TOF-MS analysis of the amygdalin produced by 5. amy/o/sentence Ba-BPD1 according to Example 7 of the present invention. In Fig. 2, the molecular weights of the abundance homologues were identified as 1449, 1463, 1477, 1491 and 1505Da, and these abundance homologs and Ba-BPD1 were used in the food industry and agriculture. Example 8: Surfactant with oysters Surfactin is a bacterial cyclic lipopeptide or surfactant that acts as an anti-201022439 biomass with its amphiphilic properties that help the substance to be present in hydrophobic and hydrophilic Two environments. For example, surfactin can be expressed in milk against Escherichia coli (^^心(/) (7) antibacterial activity ^ 乂 sterilization of milk (1) ^ ... ^ 哗 et al (2008) torsion surface activin has diesel Contaminated water and soil biodegradability (2Q). Therefore, surfactant can be used as a fungicide for food manufacturing and food preservation, and as a surfactant (or bio-degradation and removal of biodegradation and removal in industry, agriculture, and the environment). Surfactant). In order to prove that Ba-BPD1 can produce surfactant, φ large-scale and c/ms Ba-BPD1 bacterial liquid and surfactant are prepared as follows. Culture at 30 ° C, 200 rpm and Ba-BPDl After 16 hours, the cultured medium was inoculated in Cooper's medium at a ratio of 1/100 and cultured for 120 hours at 3 ° C. Cooper, s medium was mineral salts with 4% glucose (mineral salts, containing 〇.〇5 Μ NH4N03, 0.03 Μ ΚΗ2Ρ04, 〇.〇4 Μ Na2HP04, 8.〇χ ΚΓ4 Μ MgS04, 7.〇χ ΙΟ·6 Μ CaCl2, 4. 〇χ Μ6 Μ FeS04 and 4.〇χ 10 Μ Na;^ ethylenediaminetetraaeetie Add (Να2 HDTA» 〇 〜~ remove the body, add The concentrated hydrochloric acid was added to the culture solution of Ba_BpDi φ to separate the natural surfactant. The collected, dried and extracted with dichloromethane were used to form a precipitate in ρΗ2. The dichloromethane was removed under reduced pressure to obtain a yellow-white solid. The recrystallization was further purified. The dichloro, alkane extract was dissolved in sterile water containing sufficient sodium hydroxide to reach pH 8. This solution was further filtered on Whatman No. 1 filter paper and titrated to pH 2 with concentrated hydrochloric acid. A white solid precipitate. Further, the refined surfactant was purchased from Sigma (steinheim, Germany) or purified from Bacillus subtilis as a calibration standard. The isolated surfactant extract was dissolved in 1 ml of sterol. It was treated with activated carbon and filtered through a 0.22 μm pore size filter paper, and the filtrate was injected into a reverse column (rp_i 8, 5 μιη, 4 x 250 mm; Merck) of a high performance liquid chromatography apparatus 17 201022439 (HPLC). The column was eluted with 3,8 mM acetonitrile-trifluoroacetic acid (80:20 (v/v)) at a flow rate of 1.0 ml/min and detected at a wavelength of 210 nm. Sigma company bought the fine The chain surfactant was measured as a calibration curve, and the total amount of the six surfactant anions was used as the rolling degree of the surfactant. The experiment proved that: 5. amy / o / zXacie / is Ba-BPD 1 produced a surfactant concentration of 460 mg / L ° See Figure 1, according to the embodiment of the case 8, by and amy / o ^ qiiefadens φ Ba - lc/TOF-MS analysis of surfactants produced by BPD1. In Figure i, the molecular weight of the surfactin homologues was identified as 1〇22 and 1〇36 Da. Therefore, the surfactant produced by Ba-BPD1 can be used for food sterilization, food preservation, biodegradation, and industrial, agricultural, and environmental removal.

According to the results of Examples 7 and 8, it was found that the three lipopeptides - Isophyrin A, Adiponectin and Surfactant can inhibit the growth of pathogenic fungi and pathogenic bacteria. In order to determine the ability of antagonistic pathogenic fungi that is ugly like Ba-BPD1, the φ culture φ and the test procedure are as follows. The sputum test showed inhibition of growth of one organism to another. Cultured and α/πΜχβα·starvation Ba-BPD1 and 21 fungi. A single colony of quarantine/Me like Ba-BPD1 was inoculated in 5 ml of LB medium and cultured at 30.00 rpm for 7 days. A 1 cm diameter fungus (mycelial disc) was placed in the center of each of the potato dextrose agar (PDA) plates and cultured at 25. 〇: Until the fullness, 21 strains of fungi were cultivated. At the beginning of the sputum test, place the above i cm diameter fungal hyphae in the center of a piece of PDA medium and place 3 pieces of 9 mm diameter filter paper on pDA medium. 18 201022439 Each piece of filter paper is 1.8 cm from the edge of the fungal hyphae. The three filter papers placed in the PDA medium look like the three vertices of an equilateral triangle. On the experimental group, 30 μM of Ba-BPD1-like liquid was added dropwise to each filter paper of the PDA medium; in the control group, 30 μM of sterile water was dropped on each filter paper of the PDA medium. These PDA media were cultured at 25 ° C until they were overgrown. A crescent-shaped suppression will form around the filter paper. Records and the 峙 culture between Ba-BPD1 and the fungal strain, and calculated the distance between the square (B) and the scorpion, such as Ba-BPD1 filter paper to the fungal hyphae. ❿ Refer to Table 1 for the average enthalpy distance between the ugly.am_y/o/per-Ba-BPD1 filter paper and the fungal hyphae of Example 9 of the present case. The longer the distance between the cockroaches, the better the fungus inhibition effect. In Table 1, it can be seen that the growth of the 21 species of fungi can be effectively inhibited by Ba-BPD1, wherein 5.am3; the pair between Zo%Me/acie and Ba-BPD1 and the citrus Penicillium round 'abbreviated to pin' The longest distance (13.5 mm) and the shortest distance between Ba-BPD1 and G. mellifera (G/omere//a, abbreviated as Gc) (3.1)

Imn). Therefore, 5. The growth inhibitory effect φ of Ba-BPD1 on fungi was confirmed. Rehabilitation Example 10: and BarBPD1 and pathogenic bacteria are on the road Another 5. Ice zci'e like Ba-BPD1 and pathogenic bacteria test the following. First, 6 μl of the test to be tested and flmj/o/zXfldewBa-BPD1 were dropped on the filter paper of the nutrient medium, and cultured at 30 ° C for 24 hours. Each pathogenic bacteria to be tested was evenly sprayed onto the cultured Acacia medium containing the cultivated Ba_BPD 1, and the medium was cultured at 30 ° C for 24 hours. The pathogen fine | g was subjected to three repeated tests. Determine the size of the inhibition zone of a pathogenic bacteria. 19 201022439 Please refer to Table 2' for the inhibition circle size of pathogenic bacteria of 5. Ba-BPD1 of Example 10 of the present invention. In Table 2, it is known that Ba-BPD1 can effectively inhibit the growth of these pathogenic bacteria. Therefore, pathogenic plant and fruit diseases caused by these bacteria are prevented, treated or controlled. In addition, and amyloliquefaciens Ba-BPDl ^ (Bacillus cereus 01) and Salmonella (SaimmeUa τΑι〇()), while η amy_uefadens

Ba-BPDl can be poisoned by the finest foods that are picked up by these fine (five).

The novel liquefied Bacillus amyloliquefaciens of the invention has a unique sequence of clear nucleus corpus corpus, and can produce multi-shholesin (four) decomposition, egg and spider ' and can produce anti-biomass, and has effect on pathogenic fungi and pathogen fine ==2 effect, It is difficult to _ invention, deep production of Wei value, aid two. Further, the present invention may be modified by those skilled in the art (4) without departing from the scope of the appended claims. -❹ 20 201022439 ❹ H and, /〇/_/如娜娜Ba-BPD1 filter paper and fungus bacteria-- pathogenic fungi 3 (Botrytis eUiptica) Be 9.2 Rose ash disease (So noisy to dnerea) Be 8.8 Glomerella cingulata Gc 3.1 Colletotrichum musae Cm 9.8 Rhizoctonia solani Rs 4.0 Fusarium oixysporum t sp· pisi F307 10.5 Fusarium ojtysporum t sp· lycopersici F308 5.2 Fusarium ojsysporum f· sp· fycopersici Fol-33 7.7 Phalaenopsis elegans (Fw^ar/Mm w/izm) FSO 7.3 Fusarium solani FSL 7.5 Lirioduria fuliginea (iScfcroftww ra you/z· Saccardo) Sr 3.0 Apple brown leaf spot (zi/如7Μζη·α wmz/z·) Am 8.0 (Phytophthora capsici) PcSl 5.0 Aspergillus niger Anl2 5.0 Aspergillus niger An22 4.0 Penicillium (Penici7/iwm fia/icum) Pil3 13.5 Phase 'P. citrinum (尸 ewia7iiwm ito//cwm) Pi28 12.3 beginning. Good marks instrument {Colletotrichum gbeo^> oidoid Es) Cg-T4018 7.8 Colletoirichum gloeosporioides Cg-T4044 9.4 Pestabtiopsis eugeniae Pe 7.3 Phytophthora i. Botry odoplodia theobromae Bot 9.3 21 201022439 (Let <<<<<>)奥倒凾[·♦# η 60 ΓΓΛ S. inch S. inch ς·inch 6(ν Ι·ει·ε ε(Ν SZ sfnετ inch·ε, f, Y 豳【♦ 客客堀田r^€^ laPHH-eoq§apQ/asi&//o/rsw/oar,(N^(suluosod poojΊ3μ9;υΒΒ#ψ 堀田3#伞(suaosod POOJ-s-c313Bg)^-a-#^^堀J. (1 -aHq Jsl Ι.2δρΒ«Butterfly Dragon Lord (B-2ss2q jo lojags) 喋实坫蚱+- (OIBSOJ JO 10ds-sp3PBg)€?flssi®4#f★实垛(J35IUB3 snt;u)€嵘*?龙老(3usiMJ.sJ3PBg) 嗦Long porphyrin (SJOds Jsn.sJ3PBg) 嗦钹崁iiMJ.寞¢ (3SBJP-SJ y〇s) €_^MMJ.· (3SBtu.2p iojcos )€痛^iiMJ ?J IBSq l.2J3PBg)€鳗^湎凛湎凛湎凛 (113⁄4 UMOJU) 螺*土(UOPUI JO q3103.-tsSJ IBP3PBCQ) coal 龊崁21涠<1黩^ (001VISFS—3) g V L5 (I §sf §13 §==0005)堀竑钭彳^ (a^z^/O.AdaQZ'o^QWOM/o^WQ^)堀龙贼^键^ ·>λ &§so^ Sa^ 0 (αμ5α°/δΛ ·>α ^ο §§^8 Β§εο^§^ s 050 ·>Λ ^ο^ο§Ηα B§so^§^ s (ss^auQW^oy-wo^/n^) ® 螺龙命(an^w /.i/is'SWOM/opnasy^M^ia^® B-Duohan (/sa^§svo^-2w//K^)M竑_Restaurant suffers ^soso^ao so§o^av 0 M iavsvop JofoaqoJ-u^0 铁一^ι,0Μ^^ (srsforosaEso^ng) bundled 殍渖 殍渖 迩 迩 ^ ^ ( ( ( ( ( ( ( ( ( ( ( ( ( (i§p^aw/5 s·21'^00·00.^^ )涵^灿(///sip.dsqns am/aAS ΧΤΜΟΛΟΡΡ') s 镙 堀 堀 堀 ^ τι 201022439 [Simple description of the diagram] Figure 1 is based on the case of the case 7 and 8, by ugly

LC/TOF-MS analysis of isincin a and surfactants produced by Ba-BPD1. Fig. 2 is an LC/TOF-MS analysis map of amylin produced by ugly awd/o^w^/^dewBa-BPD1 according to Example 7 of the present invention. [Key component symbol description] No reference: 1. Ahmed, TAE, Dare, EV and Hincke, M. Fibrin: a versatile scaffold for tissue engineering applications. Tissue Eng. Part B Rev. 2008. 14(2): 199- 215. 2. Alam, Μ. Z., Muyibi, SA and Wahid, R. Statistical optimization of process conditions for cellulase production by liquid state bioconversion of domestic wastewater sludge. Bioresour. Technol. 2008. 99(11): 4709-16 3. Bodour, AA, Drees, KP and Maier, RM Distribution of biosurfactant-producing bacteria in undisturbed and contaminated arid southwestern soils. Appl. Environ. Microbiol. 2003. 69(6): 3280-7. 4. Deleu, M ., Paquot, M. and Nylander, T. Effect of fengycin, a lipopeptide produced by Bacillus subtilis, on model biomembranes. Biophys. J. 2008. 94(7): 2667-79. 5. Drouin, M., Lai, CK, Tyagi, RD and Surampalli, RY Bacillus licheniformis proteases as high value added products from fermentation of wastewater sludge: pre-treatment of sludge to increase t Water performance at the process. Water Sci. Technol. 2008. 23 201022439 57(3): 423-9. 6. Elsheikh, L, E., Bergman, R., Cryz, SJ Jr. and Wretlind, B. A comparison Of different methods for determining elastase activity of Pseudomonas aeruginosa strains from mink. Acta Pathol. Microbiol. Immunol. Scand. Sect. B, Microbiol. 1986. 94(3): 135-8. 7. Emtiazi, G., Naghavi, N And Bordbar, A. Biodegradation of lignocellulosic waste by Aspergillus terreus. Biodegradation, 2001. 12(4):257-61.

8. Ertugrul, S., Donmez, G. and Takag, S. Isolation of lipase producing Bacillus sp. from olive mill wastewater and improving its enzyme activity. J. Hazard. Mater. 2007. 149(3): 720-4. 9. Hsieh, F.-C., Lin, T.-C., Meng, M. and Kao, S.-S. Comparing methods for identifying Bacillus strains capable of producing the antifungal lipopeptide iturin A. Curr. Microbiol. 2008 56(1): 1-5. 10. Hua, Y., Jiang, B., Mine, Y. and Mu, W. Purification and characterization of a novel fibrinolytic enzyme from Bacillus sp. nov. SK006 isolated from an Asian Traditional fermented shrimp paste. J. Agric. Food Chem. 2008. 56(4):1451-7. 11. Huang, X., Wei, Z., Zhao, G., Gao, X., Yang, S. and Cui, Y. Optimization of Sterilization of Escherichia coli in milk by surfactin and fengycin using a response surface method. Curr. Microbiol. 2008. 56(4): 376-81. 12. Ito, S., Kobayashi, T., Ara , K., Ozaki, K., Kawai, S. and Hatada, Y. Alkaline detergent enzymes from alkaliphiles: enzymatic properties, genetics, and struc Tures. Extremophiles. 1998. 2(3): 185-90. 13. Jeganathan, J., Nakhla, G and Bassi, A. Hydrolytic pretreatment of oily wastewater by immobilized lipase. J. Hazard. Mater. 2007. 145(1 -2): 127-35. 24 201022439 14. Kumar, A. G, Swamalatha, S., Sairam, B. and Sekaran, G. Production of alkaline protease by Pseudomonas aeruginosa using proteinaceous solid waste generated from leather manufacturing industries. Bioresour Technol. 2008. 99(6): 1939-44. 15. Lanciotti, R., Gianotti, A., Baldi, D., Angrisani, R., Suzzi, G., Mastrocola, D. and Guerzoni, Μ. E. Use of Yarrowia lipolytica strains for the treatment of olive mill wastewater. Bioresour. Technol. 2005. 96(3): 317-22. 16. Mandel, M. and Reese, ET Induction of cellulose in fungi by cellobiose. J. Bact. 1960. 79: 816-26.

17. Mizumoto, S. and Shoda, M. Medium optimization of antifungal lipopeptide, iturin A, production by Bacillus subtilis in solid-state fermentation by response surface methodology. Appl. Microbiol. Biotechnol. 2007. 76(1): 101-8 ~ 18. Romero, D., de Vicente, A., Rakotoaly, RH, Dufour, SE, Veening, J.-W., Arrebola, E., Cazorla, FM, Kuipers, OP, Paquot, M. and Perez -Garcia, A. The iturin and fengycin families of lipopeptides are key factors in antagonism of universal quotient"/& toward Podosphaera fusca. Mol. Plant Microbe Interact. 2007. 20(4): 430-40. 19. Sangave , PC and Pandit, AB Enhancement in biodegradability of distillery wastewater using enzymatic pretreatment. J. Environ. Manage. 2006.78(1): 77-85. 20. Whang, L.-M., Liu, P.-W. G, Ma, C.-C. and Cheng, S.-S. Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil. J. Hazard. Mater. 2008. 151(1): 155- 63. 25

201022439 Sequence Listing <110> Executive Yuan Agricultural Committee Agricultural Drug Toxicology Laboratory <12〇> Novel L. liquefaciens strain and its application <160>1 <210>1 <211>1421

cggcuaccac uuacagaugg 1 caagucgagc ggacagaugg gagcuugcuc ccugauguua gcggcggacg ggugaguaac acguggguaa ccugccugua agacugggau aacuccggga aaccggggcu aauaccggau gguuguuuga accgcauggu ucagacauaa aagguggcuu; < 212 > RNA < 213 > Bacillus amylolicjuefaciens Ba-BPDl < 220 > < 223 > PARTIAL 16S RIBOSOMAL RNA SEQUENCE < 400 & gt acccgcggcg cauuagcuag uuggugaggu aacggcucac caaggcgacg augcguagcc gaccugagag ggugaucggc cacacuggga cugagacacg gcccagacuc cuacgggagg cagcaguagg gaaucuuccg caauggacga aagucugacg gagcaacgcc gcgugaguga ugaagguuuu cggaucguaa agcucuguug uuagggaaga acaagugccg uucaaauagg gcggcaccuu gacgguaccu aaccagaaag ccacggcuaa cuacgugcca gcagccgcgg uaauacguag guggcaagcg uuguccggaa uuauugggcg uaaagggcuc gcaggcgguu ucuuaagucu gaugugaaag cccccggcuc aaccggggag ggucauugga aacuggggaa cuugagugca gaagaggaga guggaauucc acguguagcg gugaaaugcg uagagaugug gaggaacacc aguggcgaag Gcgacucucu ggucuguaac ugacgcugag gagcgaaagc guggggagcg aacagg in uua gauacccugg uaguccacgc cgu ugly ugly cgau gagugcuaag u guuaggggg uuuccgcccc uuagugcugc agcuaacgca uuaagcacuc cgccugggga guacggucgc aagacugaaa cucaaaggaa uugacggggg cccgcacaag cgguggagca ugugguuuaa uucgaagcaa cgcgaagaac cuuaccaggu cuugacaucc ucugacaauc cuagagauag gacguccccu ucgggggcag agugacaggu ggugcauggu ugucgucagc ucgugucgug agauguuggg uuaagucccg caacgagcgc aacccuugau cuuaguugcc agcauucagu ugggcacucu aaggugacug ccggugacaa accggaggaa gguggggaug acgucaaauc aucaugcccc uuaugaccug ggcuacacac gugcuacaau ggacagaaca aagggcagcg aaaccgcgag guuaagccaa ucccacaaau cuguucucag uucggaucgc agucugcaac ucgacugcgu Gaagcuggaa ucgcuaguaa ucgcggauca gcaugccgcg gugaauacgu ucccgggccu uguacacacc gcccgucaca ccacgagagu uuguaacacc cgaagucggu gagguaaccu uuuaggagcc agccgccgaa g 1421 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 26 1380

Claims (1)

201022439 * X. Patent application scope: L An isolated strain comprising a 16S ribosomal RNA comprising at least a partial nucleotide sequence of SEQ ID NO: 1, or 98% or 98% or more A sequence similar to SEQ ID NO: 1. 2. The strain according to claim 1, wherein the strain belongs to a liquefied starch spore and a kiss (c/ms). 3. The strain according to claim 1, wherein the strain is a liquefied strain of Bacillus granulosus Ba-BPD1, and the patent micro-stock registration number is BCRC 910395, or a mutation having all the identification characteristics of the strain. Strain. 4. A use according to the strain of the first application of the patent scope, which is for producing at least one enzyme in the following group consisting of: amylolytic enzyme, proteolytic enzyme, cellulolytic enzyme and lipid decomposition Enzyme. 5. The species of the first g of the first strain, such as the 14th, is decomposed into at least one of the following organic components, including: white matter, cellulose, lipids and blood clots. • The use of strain No. 1 for the treatment of wastewater, tract systems and organic waste. 7. Add = the use of the strain called the first strain of the patent scope, which is used for the animal feed. 8. The use of the first strain of the patent scope is as follows. Use, which is used to produce the following group of active and abundance components, including: avermectin, surface 27 201022439 10. The use of the strain of the first category of the patent application, which is used For the production of biosurfactants. 11. Use of a strain according to the first aspect of the patent application for the prevention or treatment of a plant, fruit or animal infected by a fungal or bacterial infection. 12. The use of claim 9, wherein the fungal infection is caused by at least one fungus of the following group consisting of: Botrytis elliptica, politic ash, disease (Botrytis cinerea), plague disease (Glomerella cingulatd), fragrant sputum 蛰❹ "1 milk from", sweet persimmon anthracnose ((1: 〇 / 0 furnace & / ^ tolerance / 〇 6〇 5^(^/〇/£^5), Rhizoctonia so 丨ani, Fusarium oxysporum t sp./wW, F. sp. Fantasy 'c/), Fusarium solcini, Fusarium solani, Acne bacillus, Sc/m, such as m to ϊ Saccardo, apple brown spot, Phytophthora capsici, artichoke black, Aspergillus m'ger, Penicillium citrinum (Pe«icz7/z·zYWcww), Pestalotiopsis eugeniae a rot (B〇try〇dipl〇dia φ 汍eMraw); and the bacterial infection is caused by at least one of the following group consisting of: bacteria Soft rot fungi (five chrysanthemi; Erwinia carotovora subsp. carotovora ) > sex spot bacteria (icWovonxc ave (10) e subsp. citruHi), cancerous bacteria), carnation flower, Burholderia caryophylli, cockroach white bacillary rot (Enterobactor c/oflceae), carambola bacterial spot bacteria (Psewcfomoms·, Ralstonia > Si)/imacearMw, X. sinensis (Xowi/iomoww αχο-ocfc pv. cz>"), Solanaceae Plant bacterial spotted bacteria plus swelling "os 28 201022439 also ρν·, cruciferous black rot fungi (χ (10) gift campestris pv. compestris) and rice bacterial blight (xanth〇m〇nas oryzae ρν·, cactus bacillus (Bacz7/ MiS cerew <y) and Salmonella 0 13. The use of the strain of the first category of the patent application is for an antimicrobial agent. 14. The use of claim 11, wherein the antimicrobial agent inhibits growth of at least one fungus in the group consisting of: Botrytis elliptica, a rule Gray count disease* (fiotrytis cinerea), Glomerella cingulata, Colletotrichum musae, C〇netotrichum gloeosiporioides, Rice Sl. <2«z·), Phytophthora sojae (F(10)ar/ww f. sp./?/<?/), F. sylvestris (Fwsarzww f. sp. (ycopersicz·), cyanobacteria So/am), Phytophthora (Dalga/7-face, Sclerotium rolfsii Saeeardo, Alternaria mali, Pfytophthora capsici, artichoke black Bacteria (Aspergillus φ Wger), Penicillium lucidum ζΥα/icwm), Pestalotiopsis eugeniae, Botryodipbdia; and the antimicrobial agent inhibits growth of at least one of the following constituent groups The composition group includes: bacterial soft rot fungus (five rvWm*a chrysanthemi; Erwi Nia carotovora subsp. carotovora ) 'gc spotted bacteria (Jc/i/ovorox ovewae subsp. citrulli), cancer disease iww light Wmy), carnation family flower bacterium (Burholderia caryophylli), white lenticular base rot Instrument (Enterobactor·c/oacefle), Phytophthora sphaeroides (/^ewifomonos, bacterial wilt 29 201022439 (Ralstonia solanacearum), pine ulcer window (xanfj2〇moms ρν· czW), $ plant bacterial spots Bacteria (3) Training (10) (10) Melon and ρν. να/α^π·α), Cruciferous black rot fungus (also „加膨·从烈烈冲pV.(3)mpe你与稻白叶栝菌江(10) Plus foot face oryzae Pv ^Nove), Cactus bacillus (plus c///(10)(10)) and Salmonella {Salmonella) 〇15. Use as described in Sections 4, 5, 6, 7, 8, 9, 10, 11 or 13 of the patent application 'The type of this strain is used for whole liquid culture, supernatant, wettable powder, granules, water-dispersible granules, aqueous suspension and microcapsule type. 16. A mutant strain of a strain according to claim 1, wherein the mutant has a 16S ribosomal RNA sequence of SEQ ID NO: 1. A composition comprising a strain of Bacillus licheniformis (BaczY/tw am/o/z々w^/bciem· Ba-BPD1), wherein the Bacillus algolyticus strain has the patented microbial accession number BCRC 910395. ❿ 30